Deciphering the complex interactions of the various components of the insulin-like growth factor (IGF) system [IGF-I and -II peptides, type I and II IGF receptors, and IGF-binding proteins (IGFBPs)] is important for our understanding of cell growth regulation. We report here that IGF-II can enhance IGF-I-stimulated cell proliferation independent of direct IGF-II interaction with type I or II IGF receptors. Human fibroblasts cultured in serum-free medium for 40 h were relatively resistant to the mitogenic effects of added IGF-I. However, preexposure of the cultures to low concentrations of IGF-II enhanced IGF-I action several-fold. IGF-II by itself had no stimulatory effect and did not influence [Gln3,Ala4,Tyr15,Leu16]IGF-I or insulin-stimulated DNA synthesis. IGF-II did not directly interact with type I IGF receptors, as [Leu27]IGF-II, an IGF-II analog that does not bind type I IGF receptors, could mimic IGF-II's potentiating effect. Type II IGF receptors also were not involved because 1) [Gln6,Ala7,Tyr18,Leu19,Leu27]IGF-II, an analog with normal receptor binding, had no effect; and 2) beta-galactosidase, a competitive inhibitor of IGF-II receptor binding, did not influence IGF-II potentiation of IGF-I action. Enhanced cell responsiveness to IGF-I appears to be due to IGF-II-induced changes in pericellular IGFBP-3 and IGFBP-4. These data support the hypothesis that IGF-II can potentiate the action of IGF-I by disrupting the IGFBP barrier at the cell surface, thereby increasing IGF-I availability for type I IGF receptor interaction.